The present invention relates to an immunoassay or immunological determination process for antigens, haptens or antibodies, which is more particularly usable for the assaying of plasma proteins, such as lipoproteins.
For a number of years, lipoprotein assaying has developed in order to provide a better evaluation of the existence of a dyslipemia and in particular cardiovascular risks linked with hyperlipemias.
With this aim in mind, the total lipids (triglycerides and cholesterol) circulating in the blood were firstly assayed, but epidemiological studies very quickly revealed that the measurement of these parameters was imprecise. Interest was then attached to certain fractions of these lipids carried by particular apoproteins and, for example, HDL cholesterol and LDL cholesterol were assayed, because they may give more precise information regarding the existence of a dyslipemia.
Lipoproteins are complex molecular compounds of lipids and proteins. One of their essential features is that of having a lower density than the other serum proteins, which makes it possible to separate them from the other proteins and even to classify them by ultracentrifugation.
Thus, these lipoproteins can be classified according to their density and the following fractions can be separated:
(1) chylomicrons with a density below 0.94 g/l, which are the largest and least dense particles consisting of approximately 98% lipids; PA1 (2) very low density lipoproteins with a density of 0.94 to 1.006; PA1 (3) low density lipoproteins with a density of 1.006 to 1.063; PA1 (4) high density lipoproteins with a density of 1.063 to 1.21; PA1 (5) very high density lipoproteins. PA1 (1) contacting a sample containing the substance to be assayed with a labelled immunoactive reagent specific to said substance, the immunoactive reagent quantity being such that the substance to be assayed is in excess compared with the labelled immunoactive reagent; PA1 (2) contacting at least part of the thus obtained reaction medium with a solid phase, to which is fixed an immunoactive reagent specific to the substance to be assayed; PA1 (3) separating the reaction medium from the solid phase; and PA1 (4) determining the labelled immunoactive reagent content of the solid phase. PA1 (1) contacting a blood plasma sample containing the lipoproteins to be assayed with a conjugate compound of an antibody specific to said lipoproteins and an enzyme in quantities such that the lipoproteins to be assayed are in excess compared with the antibody; PA1 (2) contacting at least part of the thus obtained reaction medium with a solid phase to which is fixed the same antibody specific to the lipoproteins to be assayed; PA1 (3) separating the reaction medium from the solid phase; and PA1 (4) determining the enzyme content of the solid phase. PA1 a system of tubes, each having a solid phase, e.g. a bladed device, coated under the same conditions with human antilipoprotein immunoglobulins, said tubes being kept protected from moisture in adequate blister-type packs; PA1 a series of bottles containing standard samples covering the lipoprotein range 0.1 to 0.005 g/l, which can be stored in liquid form with the addition of adequate stabilizers, such as antibiotics, antioxidants and a protease inhibitor, or in lyophilization form, lyophilization being carried out in the presence of sucrose; PA1 a bottle containing the conjugate compound resulting from the coupling of human antilipoprotein immunoglobulins with an enzyme, such as the horse-radish peroxidase, said compound being possibly prediluted in a borate buffer containing delipoproteinated sheep serum and a preservative, such as thimerosal, in liquid or lyophilized form; and PA1 a bottle containing a chromogen for enzymatic detection, said chromogen being possibly in lyophilized form and in this case the kit also has a bottle containing a chromogen dilution buffer. PA1 (1) contacting the sample containing the .alpha.-foetoprotein to be assayed with a conjugate compound of an antibody specific to the .alpha.-foetoprotein to be assayed and an enzyme, in a quantity such that the .alpha.-foetoprotein to be assayed is in excess compared with the antibody; PA1 (2) contacting at least part of the thus obtained reaction medium with a solid phase to which is fixed an antibody specific to the .alpha.-foetoprotein to be assayed; PA1 (3) separating the reaction medium from the solid phase; and PA1 (4) determining the enzyme content of the solid phase. PA1 a system of tubes each having a solid phase coated under the same conditions with human anti .alpha.-foetoprotein immunoglobulins; PA1 a series of bottles containing standard samples having the .alpha.-foetoprotein range of 50 to 1000 ng/ml; PA1 a bottle containing the conjugate compound resulting from the coupling of a monoclonal antibody specific to the .alpha.-foetoprotein with an enzyme; and PA1 a bottle containing a chromogen for enzymatic detection.
However, this empirical classification based on the -physicochemical characteristics of lipoproteins gives very heterogeneous classes based on their composition in proteins. Thus, low density lipoproteins essentially contain apoproteins B, but also apoproteins C and apoproteins E, whilst the high density lipoprotein fraction mainly contains apoproteins A.sub.1 and apoproteins A.sub.2, but also apoproteins C, apoproteins D and apoproteins E.
Interest was then attached to the part played by these apoproteins in the transport of lipids in order to obtain a better understanding of the relatively complex metabolism of these lipids. It was attempted to show that the assaying of certain of these vector proteins could be a superior indicator in connection with the detection of dyslipemias. In addition, other assaying methods were developed to more accurately determine the amount of certain apolipoproteins contained in the blood plasma.
However, these assaying methods lead to certain problems, due to the high apolipoprotein concentration of blood plasma and the limited variation in the level of the lipoproteins between so-called normal states and so-called pathological states. For example, hereinafter are given the plasma concentrations of lipoproteins and apolipoproteins in male normolipemic patients aged between 25 and 40.
______________________________________ LIPOPROTEINS APOLIPOPROTEINS mg/100 ml mg/100 ml ______________________________________ VLDL 80 Apo-B 80-100 LDL 276 Apo-AI 120-150 HDL.sub.2 86 Apo-AII 20-80 HDL.sub.3 254 Apo-E 10 HDL.sub.2 /HDL.sub.3 0.36 Apo-CI 5-10 Apo-CII 3-5 Apo-CIII 15-20 Apo-D 10-20 Apo-F 15 ______________________________________
Moreover, to obtain a satisfactory sensitivity, immmunoassay processes were developed e.g. by radial immunodiffusion, immunoturbidimetry and immunonephelometry by laser beams, as well as radioimmunology or enzymoimmunology, as described by M. Rosseneu, R. Vercaemst, K. K. Steinberg and G. R. Cooper in CLIN. CHEM. 29/3, pp. 427-433 (1983).
However, most of the hitherto developed methods have come up against difficulties making their use far from easy for the routine of a clinical biology laboratory.
Thus, immunoelectrophoresis is a difficultly automatable method. Moreover, the recommended samples (2 .mu.l) are not compatible with series assays.
Nephelometry by laser beams only requires a 1/100 dilution of the samples to be assayed, but makes it necessary to acquire specific, costly equipment. Moreover, problems are encountered when it is wished to assay samples with a very high lipid content.
Assays by radioimmunology or enzymoimmunology make it possible to obtain a good sensitivity, because they were developed for detecting very small quantities of antigens. However, they are not very suitable for assaying lipoproteins, which are present in large quantities in the blood (approximately 1 g/l). The assaying of lipoproteins by these methods makes significant dilutions of the sample necessary and these are incompatible with working in a routine analytical laboratory. Moreover, these dilutions can be the source of major errors.
In addition, an enzymoimmunoassay process for apolipoproteins B has been developed, which only requires a 200.times.dilutions of the samples. However, the stability of the reagents used in this method cannot be guaranteed beyond a few days, which makes it incompatible with routine working. This method is described in the Journal of Immunological Methods, 21, 1978, pp. 317-324.